[Chlamydomonas] Transformation of Complex I mutants

NITYA SUBRAHMANIAN via chlamy%40net.bio.net (by subrahmanian.1 from buckeyemail.osu.edu)
Fri Aug 27 11:39:42 EST 2010


 Hi everybody,

I am working on mutants in Chlamydomonas  deficient in mitochondrial Complex I activity. We are having problems in successfully transforming the nuclear dna of these mutants. Our usual method of transformation is through electroporation. The efficiency of transformation is extremely low and the few colonies that do survive the transformation procedure turn out to be false positives.

Does anybody have any suggestions to make the transformation more efficient?

The procedure of transformation that I use is as follows:

Liquid cultures in the upper log phase: 3x106 - 6x106 cells / ml are taken and the cells are pelleted and resuspended in autolysine to give a final concentration of 108 cells/ml. After treating with autolysine for 2.5 - 3 hrs, the cells are pelleted and resuspended in minimum media + 40mM sucrose to again give a final concentration of 108 cells/ml. Then 250 microlitres of this suspension is used for transformation with sperm dna and the required amount of plasmid. Electroporation is done at 1.3kV and the pulse rate is usually 2.6-3 ms. After transformation, the cells are resuspended in 3ml TARG liquid and recovered overnight in low light. The next day, they are spread on the solid selection media and the plates are kept in high light.


Thank you very much.
Nitya.



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